抗大肠杆菌的新型溶菌酶的设计及表征

Design and characterization of a novel lysozyme against Escherichia coli

大肠杆菌是常见致病菌,对多种抗生素已产生耐药性,因此,需要开发新型抗菌剂用于预防及治疗大肠杆菌感染.然而,大肠杆菌独特的细胞外膜结构可阻止多种抗菌剂进入菌体发挥作用.为提高抗菌剂的跨膜杀菌性能,选取T4噬菌体编码的T4溶菌酶,在其N端融合不同种类的细胞穿透肽,并对融合蛋白进行重组表达.细胞壁降解实验及碘化丙啶染色实验表明,融合了细胞穿透肽的T4溶菌酶保留了其催化活性,同时具有穿膜特性.杀菌实验证明,阳离子型细胞穿透肽Pc及两亲型细胞穿透肽Pa与T4溶菌酶的融合可极大提高酶对大肠杆菌的跨膜杀菌活性,且低浓度EDTA的辅助可进一步提高融合酶的杀菌效果.其中,对于初始菌浓为10^(6) CFU/mL的Top10菌株,联合使用0.35μg/mL的Pa-T4L融合酶与0.3 mM EDTA,可在室温条件下于3 h内杀灭94%的菌体.研究结果可为针对革兰氏阴性菌的抗菌剂设计提供新思路.

Escherichia coli is a common pathogen that has acquired tolerance to a wide range of antibiotics.Thus,it is necessary to develop novel antimicrobials for the prevention or treatment of E.coli infections.However,many of these antimicrobials are inactive against E.coli due to the presence of its outer membrane serving as a permeation barrier.To enhance the outer membrane-penetrating capability,T4 lysozyme(T4L)encoded by T4 phage was selected for enzyme engineering in this study.Various cell-penetrating peptides(CPPs)were fused at the N-terminus of T4L,and the fusion enzymes were recombinantly expressed.These fusion enzymes retained the native activity of T4L and acquired membrane-penetrating capability according to cell wall-degradation analysis and cell staining by propidium iodide.Cell killing assay demonstrated that the fusion of a cationic CPP Pc or an amphipathic CPP Pa to T4L greatly improved the cell lytic activity of T4L against intact E.coli cells;in addition,the supplementation of EDTA at a low concentration could further enhance cell killing.Among all the fusion enzyme constructs,Pa-T4L at 0.35μg/mL together with 0.3 mM EDTA could kill 94%of 10^(6) CFU/mL E.coli Top10 cells at room temperature within 3 h.This study will provide guidance to the design of novel antimicrobial enzymes towards Gram-negative bacteria.